Derivatized biotin compounds and methods of use

ABSTRACT

An efficient synthesis for the selective and efficient 1′-N derivatization of biotin is reported. The derivatized biotin acts as a stable analog of the carboxyphosphate intermediate in naturally-occurring biotin-mediated CO 2  transfer. The synthesis may readily be scaled up to perform large-scale, selective acylations of biotin. The stable analog of the intermediate can inhibit the activity of the biotin carboxylase enzymes such as acetyl CoA carboxylase, and HIV protease. The functionalization at the 1′-N of biotin results in the attachment of an electrophilic “handle” amenable to reaction with a wide variety of nucleophiles to generate a new family of biotin analogs.

This is a divisional of application Ser. No. 09/321,039, filed May 27,1999, now U.S. Pat. No. 6,242,610, issued Jun. 5, 2001.

The development of this invention was funded in part by the Governmentunder cooperative grant number GM51261 awarded by the NationalInstitutes of Health. The Government has certain rights in thisinvention.

This invention pertains to derivatized biotin compounds, and to methodsof using those compounds.

Biotin (also known as vitamin H) is an essential growth factor found inall animals, plants, fungi, and bacteria. It is found, for example,bound to proteins or polypeptides in the liver, pancreas, kidney, milk,and in yeasts. Biotin is a cofactor for a group of enzymes that catalyzecarboxylation reactions, transcarboxylation reactions, anddecarboxylation reactions. The reactions catalyzed by biotin-dependentenzymes are involved in several essential metabolic pathways, includinggluconeogenesis, fatty acid synthesis, and amino acid catabolism. Allbiotin-dependent carboxylases catalyze their respective reactions viathe two step reaction shown below:

Enzyme-biotin+Mg²⁺+ATP+HCO₃ ⁻⇄Enzyme-biotin-CO₂ ⁻+Mg²⁺+ADP+Pi  (1)

Enzyme-biotin-CO₂ ⁻+acceptor⇄acceptor-CO₂ ⁻+Enzyme-biotin  (2)

R. Guchhait et al., J. Biol. Chem. vol. 249, pp. 6646-6656 (1974) showedthat in the first partial reaction shown above, biotin is carboxylatedon the 1′-N. Since bicarbonate is the source of CO₂ for allbiotin-dependent carboxylases, carboxylation of the 1′-N of biotin isaccomplished by activating bicarbonate through phosphorylation with ATPto form a carboxyphosphate intermediate. The carboxyl group is thentransferred from the carboxyphosphate intermediate to biotin to formcarboxybiotin.

In the field of biotechnology, biotin has become an important reagent inmethods to label, detect, and purify proteins and nucleic acids. Thesemethods are based on the remarkable affinity between biotin and theproteins avidin and streptavidin. The dissociation constant of biotinfrom avidin or streptavidin is about 10⁻¹⁵M, one of the strongest knowninteractions between a protein and a ligand. While all parts of thebiotin molecule contribute to this tight binding, hydrogen bondingdonation by the ureido nitrogens is the major contributor.

Q. Han et al., “Synthesis of (+)-Biotin Derivatives as HIV-1 ProteaseInhibitors,” Bioorg. & Med. Chem., vol. 6, pp. 1371-1374 (1996) reportedthe synthesis of several bis-N-alkylated biotin derivatives, and theiractivity against HIV-1 protease. All biotin derivatives reported in thispaper were substituted on both urea nitrogens. There was no suggestionto selectively substitute biotin at one of the nitrogen atoms only, norwas there any suggestion of how such a selective synthesis might beperformed.

Up to 25% diacylated biotin products have previously been reportedfollowing acylation of biotin methyl ester with (1) methylchloroformateor (2) trifluoroacetic anhydride. See (1) J. Knappe et al., BiochemisheZeitschrift, vol. 335, pp. 168-176 (1961); and (2) A. Berkessel et al.,Bioorganic Chem., vol. 14, pp. 249-261 (1986); respectively.

P. Tipton et al., “Catalytic Mechanism of Biotin Carboxylase:Steady-State Kinetic Investigations,” Biochemistry, vol. 27, pp.4317-4325 (1988) reported that no biotin analog that had been tested hadinhibited biotin carboxylase (at page 4322).

The ureido ring of biotin is of great importance in binding to avidin,and the 1′-N of that ring is directly involved in biotin's role as acarboxytransfer intermediate. However, there have been relatively fewstudies involving functionalization of the 1′-N of biotin. Most priorresearch on derivatizing biotin has involved acylation chemistry at thecarboxylic acid terminus of the pendant alkyl chain.

We have discovered a method to functionalize biotin at the 1′-N,selectively and with high efficiency. The resulting 1′-N-substitutedbiotin has an electrophilic “handle” that is amenable to reaction with awide variety of nucleophiles to generate a new family of biotin analogs.Such nucleophiles might include, for example, acetyl coenzyme A, benzylalcohol, benzoic acid, aniline, or other nucleophiles known in the art.

This synthesis is more selective for functionalization at the 1′-N ofbiotin than have been any known prior syntheses. Compounds prepared fromthe 1′-N-substituted biotin are useful in inhibiting various enzymaticreactions, including the inhibition of HIV protease.

As initial examples, we have synthesized compounds 1 and 3 (see FIGS. 1and 2). Compound 1 is formally derived from phosphonoacetic acid coupledto the 1′-N of biotin. The synthesis of Compound 3 was highly efficientand selective: a 98% yield with essentially 100% selectivity, in asynthesis conducted on the multi-gram scale. The synthesis of Compound 1had an overall yield of 35%, and a 100% selectivity.

We have shown that compound 1 acts as a stable analog of thecarboxyphosphate intermediate in naturally-occurring biotin-mediated CO₂transfer. Compound 1 inhibits the activity of the biotin carboxylasecomponent of the enzyme acetyl CoA carboxylase. This is the firstreported biotin-derived inhibitor of biotin carboxylase.

The synthesis of Compound 3 may readily be scaled up to performlarge-scale, selective acylations of biotin to form Compound 1 or otherend products.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts target compound 1.

FIG. 2 depicts the overall synthetic scheme for the synthesis ofcompounds 1, 3, and various intermediates.

FIG. 3 depicts the activity of a biotin carboxylase in the absence ofcompound 1, and in the presence of increasing concentrations of compound1.

MATERIALS AND METHODS

Metal-chelating, histidine-binding resin was purchased from Novagen.Pyruvate kinase was purchased from Boehringer Mannheim. All otherreagents were purchased from Sigma or Aldrich. Unless otherwise noted,all nonaqueous reactions were carried out under a dry nitrogenatmosphere in flame-dried glassware. CH₂Cl₂ was distilled over CaH₂immediately prior to use. DMF was purchased from Aldrich in sure/sealbottles and was used without further purification. Analytical thin-layerchromatography (TLC) was performed with general purpose 60-Å silica gelon glass (Aldrich). TLC plates were visualized with aqueous KMnO₄. Flashchromatography columns were prepared with Kieselgel 60-Å silica gel,230-400 mesh (Merck). Proton (¹H), Carbon (¹³C), and Phosphorus (³¹P) Nspectra were measured on a Bruker ARX300 300 MHz spectrometer. IRspectra were measured on a Perkin Elmer 1760X FT-IR spectrometer. Massspectrometry was provided by the Washington University Mass SpectrometryResource.

Phenylmethyl 5-(6,8-Diaza-oxo-3-thiabicyclo[3.3.0]oct-2-yl) Pentanoate(Compound 2)

The synthesis of compound 1 began by protecting (+)-biotin as thecorresponding benzyl ester in 95% yield by stirring a DMF solution (60ml) of biotin, (10.2 g, 42.6 mmol, 1 equiv), benzyl alcohol (5.6 g, 52mmol, 1.2. equiv), DCC (46.2 ml of a 1 M solution in CH₂Cl₂, 46.2 mmol,1.1 equi), DMAP (0.51 g, 4.2 mmol, 0.1 equiv), HOBT (0.64 g, 4.2 mmol,0.1 equiv), at room temperature for 12 hours.

More particularly, (+)-Biotin (10.20 g, 42 mmol), DMAP (0.51 g, 4.2mmol), HOBT (0.64 g, 4.2 mmol) and benzyl alcohol (5.64 g, 52 mmol) wereadded to 60 ml of DMF in a 500 ml, 3-neck flask. The mixture was stirredand heated until a homogenous solution was obtained. DCC (46.2 ml of a1M solution in CH₂Cl₂) was added over 10 min, and the reaction mixturewas stirred 12 hours at room temperature. The reaction mixture wasfiltered and the filtrate concentrated in vacuo. The resulting solid wasdissolved in hot acetone (50° C.) and purified by flash chromatographywith 10% MeOH/90% EtOAc to produce a white solid, compound 2 (13.3 g,95% yield): m.p. 79° C.; ¹H NMR (DMSO-d₆) δ7.36 (s, 5H), 6.43 (s, 1H),6.36, (s, 1H), 5.08, (s, 2H), 4.29, (m, 1H), 4.13, (m,1H), 3.07, (m,1H), 2.84-2.50, (m, 2H), 2.36, (t, J=7.2 Hz, 2H), 1.65-1.25, (m, 6H);¹³C (DMSO-d₆) δ173.59, 163.56, 137.15, 129.30, 128.79, 66.18, 61.88,60.03, 56.20, 34.15, 28.84, 25.37; IR (KBr) 3244, 2930, 1740, 1692,1458, 1262, 1170, 737, 698; HRMS: found 335.1432 (C₁₇H₂₃N₂O₃S=335.1429MH⁺).

Phenylmethyl5-(6,8-diaza-6-(2-chloroacetyl)-7-oxo-3-thiabicyclo[3.3.0]oct-2-yl)pentanoate(Compound 3)

Chloride 3 was prepared in 98% yield by careful dropwise addition ofEt₃N (7.06 ml, 5.1 mmol, 3 equiv) and chloroacetylchloride (1.95 ml, 25mmol, 1.5 equiv) in three portions to a solution of biotin benzyl ester(5.6 g, 17 mmol, 1 equiv) in CH₂Cl₂ at −78° C., followed by warming toroom temperature and holding for 12 hours.

More particularly, (5.6 g, 17 mmol) of compound 2 and (7.06 ml, 51 mmol)Et₃N were added to 40 ml of CH₂Cl₂. Chloroacetylchloride (1.95 ml, 25.2mmol) was added in three portions. The mixture was stirred and broughtto −78° C. and (0.65 ml, 8.4 mmol) of chloroacetylchloride was added.The mixture was stirred for 12 hours and allowed to warm to roomtemperature. The same series of steps was repeated 2 more times. Thereaction mixture was filtered, and the filtrate was evaporated in vacuoto a thick oil, which was then dissolved in CH₂Cl₂ and purified by flashchromatography with 4% MeOH/48% hexane/48% EtOAc to afford a yellowishoil 3 (6.9 g, 98% yield); ¹H NMR (DMSO-d₆) δ8.11, (s, 1H), 7.36 (s, 5H),5.08, (s, 2H), 4.81, (m, 3H), 4.19, (m, 1H), 3.20, (m, 1H), 3.03-2.83,(m, 2H), 2.37, (t, J=7.2 Hz, 2H), 1.71-1.23, (m, 6H); ¹³C NMR (DMSO-d₆)δ173.56, 166.32, 156.64, 137.14, 129.14, 128.80, 66.20, 62.20, 58.84,55.42, 44.89, 38.03, 34.10, 28.81, 25.24; IR (thin film) 3332, 3134,2937, 1735, 1704, 1395, 1358, 1241, 792, 752, 699; HRMS: found 411.(C₁₉H₂₃ClO₄N₂S=411.1245 MH⁺).

No diacylated product was observed. Up to 25% diacylated product hasbeen reported upon acylation of biotin methyl ester with (1)methylchloroformate or (2) trifluoroacetic anhydride. See (1) J. Knappeet al., Biochemishe Zeitschrift, vol. 335, pp. 168-176 (1961); and (2)A. Berkessel et al., Bioorganic Chem., vol. 14, pp. 249-261 (1986);respectively. In other experiments, we obtained up to 10% diacylationupon reaction of biotin methyl ester with chloroacetylchloride asevidenced by 1H NMR. Energy minimization computer studies (using Sybil™software, version 6.1) showed that the benzyl moiety blocked the 3′-Nmore effectively than would a methyl group.

Other halogens could be substituted for the chlorine atom in Compound 3.To synthesize other halogen-substituted analogs of Compound 3, one couldsubstitute other haloacetyl chlorides in the above synthesis forchloroacetyl chloride; e.g., FCH₂COCl, BrCH₂COCl, or ICH₂COCl.Substituting Br or I for Cl might make the substitution reactionsfaster. More generally, one could synthesize a phenylmethyl5-(6,8-diaza-6-(2-X-acetyl)-7-oxo-3-thiabicyclo[3.3.0]oct-2-yl)pentanoate,wherein X is a h by reacting biotin with an acid halide also containinga primary, secondary, or tertiary halide in a solvent comprising anon-nucleophilic base. The non-nucleophilic base acts as an “acidsponge” to absorb acids (such as HCl) generated in the reaction mixture,without itself participating in a nucleophilic attack. Examples of suchnon-nucleophilic bases include, e.g., triethyl amine and pyridine.

A 1′-N-substituted biotin compound may be synthesized by reacting aphenylmethyl5-(6,8-diaza-6-(2-X-acetyl)-7-oxo-3-thiabicyclo[3.3.0]oct-2-yl)pentanoatewith a nucleophile, under reaction conditions favorable to nucleophilicsubstitution, wherein X is a halogen atom.

Phenylmethyl5-(6,8-diaza-6-(2-diethoxyphosphono)acetyl)-7-oxo-3-thiabicyclo[3.3.0]oct-2-yl)pentanoate (Compound 4)

Compound 3 (6.9 g, 17 mmol, 1 equiv) underwent an Arbuzov reaction at100° C. in P(OEt)₃ solvent (20 ml) to afford the phosphonate ester 4 in89% yield. The structure of 4 was confirmed by x-ray crystallography.

More particularly, (6.88 g, 17 mmol) of compound 3 and triethylphosphite (20 ml) were added to a 100 ml, 3-neck flask and heated to100° C. with stirring for 3 hours. The excess triethyl phosphite wasevaporated in vacuo. The resulting viscous oil was dissolved in CH₂Cl₂and purified by flash chromatography using a gradient of 4% MeOH/48%hexane/48% EtOAc to 10% MeOH 90% EtOAc, to give a yellowish oil,compound 4 (7.6 g, 89%). Crystals were obtained by evaporating asolution of 4 in EtOAc; ¹H NMR (CDCl₃) δ7.28, (s, 5H), 5.04, (s, 2H),4.85, (t, 1H), 4.09, (q,J=6.9 Hz, 4H), 3.78, (m, 1H), 3.09, (m, 1H),2.94, (m, 1H), 2.29, (t, J=7.2 Hz, 2H), 1.74-1.36, (m, 6H), 1.28,(t,J=6.9 Hz, 6H); ³¹P (DMSO-d₆) δ21.3, (s); ¹³C NMR (DMSO-d₆d 173.56,165.02, 157.51, 156.60, 137.14, 129.30, 128.79, 66.20, 62.57, 62.11,60.62, 57.68, 55.46, 38.29, 34.64, 34.108, 32.92, 28.81, 28.58, 25.24;IR (thin film) 3328, 3262, 2981, 2935, 2867, 1737, 1678, 1396, 1351,1251, 1025, 753, 699; HRMS: found 513.1828 (C₂₃H₃₃N₂O₇PS=513.1824 MH⁺).Crystal data for 4: C₂₃H₃₃N₂O₇PS,monoclinic space group P2₁,a=10.546(1), b=12.550(1), c=10.674(1) Å, β=112.15(1)°, V=1308.4(5) Å³,Z=2, colorless, T=299° K., R=0.096, GOF=4.753. Displacement parametersof the benzyl ester and phosphonate groups were large; low temperaturedata collection is also planned

Phenylmethyl5-(6,8-Diaza-7-oxo-6-(2-phosphonoacetyl)-3-thiabicyclo[3.3.0] Oct-2-yl)Pentanoate (Compound 5)

Phosphonate ester hydrolysis of 4 (4.75 g, 9.3 mmol, 1 equiv) promotedby TMSBr (3.7 ml, 28 mmol, 3 equiv) afforded 3.0 g (72% yield) ofphosphonic acid 5.

More particularly, to a flask with CH₂Cl₂ (40 ml), (4.75 g, 9.26 mmol)of compound 4 were added. TMSBr (3.67 ml, 28 mmol) was added and themixture was stirred for 2 hours at room temperature. The reaction wasquenched with 5 ml of distilled water and concentrated in vacuo. Thesolid was recrystallized in water/methanol (9:1) to afford a whitesolid, compound 5. (3.03 g, yield 72%); m.p. 184° C.; ¹H NMR (DMSO-d₆)δ7.96, (s, 1H), 7.36, (s, 5H), 5.1, (s, 2H), 4.77 (t, 1H), 4.1, (m, 1H),3.5, (m, 2H), 3.2 (m, 2H), 3.07-2.80, (m, 2H), 2.37, (t, J=7.2 Hz, 2H(DMSO-d₆) δ15.5, (s); ¹³C NMR (DMSO-d₆) δ173.57, 166.40, 157.48, 156.61,129.31, 128.81. 66,20, 62.10, 57.64, 55.40, 38.47, 35.44, 34.11, 28.82,28.60, 25.25; IR (KBr) 3434, 2935, 2856, 1737, 1682, 1399, 1356, 1233,1184, 1149, 753, 698; HRMS: found 457.1194 (C₁₉H₂₅N₂O₇PS=457.1198 MH⁺).

5-(6,8-Diaza-7-oxo-6-(2-phosphonoacetyl)-3-thiabicyclo[3.3.0] Oct-2-yl)pentanoic acid (Compound 1)

Subsequent saponification of 5 (0.51 g, 1.1 mmol, 1 equiv) with LiOH(0.21 g, 4.9 mmol, 4.5 equiv) afforded the water soluble target 1 (59%yield).

More particularly, (0.21 g, 4.9 mmol) of LiOH was added to a solution(0.506 g, 0.000225 moles) of compound 5 in 40 ml of water. The reactionmixture was filtered, and the filtrate was concentrated in vacuo andrecrystallized in water/acetone (9:1) to afford a white solid, compound1. (0.209 g, yield 59%); 1H NMR (D₂O) δ4.50, (m, 1H), 4.34, (m, 1H),3.25, (m, 1H), 2.92-2.64, (m, 2H), 2.43-2.36, (m, 2H), 2.06, (t, J=7.2Hz, 2H). 1.69-1.25, (m, 6H); ³¹P (D₂O) δ14.9 (s); ¹³C NMR (D₂O) δ157.49,62.33, 60.59, 55.69, 41.38, 40.05, 39.86, 37.65, 28.59, 28.00, 25.00; IR(KBr) 3344, 3196, 2922, 2851, 1702, 1661, 1567, 1431, 1138, 1074, 869;MS: found 394.4 (C₁₂H₁₇LiN₂NaO₇PS=394.0552 MH⁺).

Purification and Assay of Biotin Carboxylase Inhibition

Compound 1 was used in enzyme inhibition studies to demonstrate that itis a reaction intermediate analog of biotin-dependent carboxylases. As aprototype example, we examined the effect of Compound 1 on the activityof biotin carboxylase from Escherichia coli. Biotin carboxylase is onecomponent of E. coli acetyl CoA carboxylase. It catalyzes the firstpartial reaction shown above as reaction (1). We chose E. coli biotincarboxylase as a model for biotin-dependent carboxylases generallybecause it was readily available—its gene has been cloned andoverexpressed. See S. Li et al., J. Biol. Chem., vol. 267, 855-863(1992); and H. Kondo et al., Proc. Natl. Acad. Sci. USA, vol. 88, pp.9730-9733 (1991). In addition, the crystal structure of biotincarboxylase has been determined, and it is the first and currently onlythree-dimensional model of a biotin-dependent carboxylase available.

Acetyl CoA carboxylase catalyzes the biotin-dependent carboxylation ofacetyl CoA to form malonyl CoA in the first step of fatty acidbiosynthesis. The E. coli acetyl CoA carboxylase has three components:(1) biotin carboxylase, which catalyzes the ATP-dependent carboxylationof biotin (step 1 in the reaction shown above), (2) the biotin carboxylcarrier protein, which contains the biotin moiety cofactor in whichbiotin is covalently linked to a lysine residue via an amide linkage tothe valeric acid carboxyl, and (3) carboxyl transferase, which catalyzesthe transfer of the carboxyl group from carboxybiotin to acetyl CoA.Acetyl CoA carboxylase in humans is a target for antihyperlipidemicdrugs. Acetyl CoA carboxylase in plants is a target for severalherbicides.

Biotin carboxylase was purified from a strain of E. coli thatoverexpresses the gene coding for the enzyme, strain BL21(DE3)pLysS,obtained from Novagen (Madison, Wis.). Purification was performed with ahistidine-tag attached to the amino terminus of a mutant form of theenzyme and nickel affinity chromatography as described in C. Blanchardet al., “Mutations at Four Active Site Residues of Biotin CarboxylaseAbolish Substrate-Induced Synergism by Biotin,” Biochemistry, vol. 38,pp. 3393-3400 (1999).

The activity of biotin carboxylase was measured spectrophotometricallyby following the production of ADP. The amount of ADP was determinedusing pyruvate kinase and lactate dehydrogenase, and the oxidation ofNADH was followed at 340 nm. Each measurement was carried out in avolume of 0.5 ml in 1 cm path length quartz cuvettes. The reactionmixture contained 10 units of pyruvate kinase, 18 units of lactatedehydrogenase, 0.5 mM phosphoenolpyruvate, 0.2 mM NADH, 8 mM MgCl₂ and100 mM HEPES at pH 8.0. For inhibition studies compound 1 was dissolvedin water, and the pH adjusted to 8 with HCl. The concentration ofcompound 1 was assayed by phosphorus analysis using the method of B.Ames et al., J. Biol. Chem., vol. 235, pp. 769-775 (1960).

We found that compound 1 inhibited the activity of biotin carboxylasesignificantly. FIG. 3 depicts the activity of biotin carboxylase in theabsence of compound 1, and in the presence of increasing concentrationsof compound 1. The substrate concentrations were in all cases heldconstant at 9 mM bicarbonate, 0.1 mM ATP, and 100 mM biotin.

The activity of biotin carboxylase decreased as the concentration ofcompound 1 increased. Preliminary characterization gave an inhibitionconstant of about 8 mM. While this value represents a modest degree ofinhibition compared to the Km for biotin in biotin carboxylase (134 mM),recall that this inhibition was achieved simply by positioning aphosphonoacetyl moiety on the 1′-N of biotin. The addition of thephosphonoacetyl moiety increased the affinity of the compound for biotincarboxylase substantially. By contrast, P. Tipton et al., “CatalyticMechanism of Biotin Carboxylase: Steady-State Kinetic Investigations,”Biochemistry, vol. 27, pp. 4317-4325 (1988) reported that no biotinanalog that had been tested had inhibited biotin carboxylase (at page4322).

Compound 3 is an electrophilic coupling template that is useful not onlyin synthesizing Compound 1, but that is useful as an intermediate insynthesizing numerous other 1′-N substituted biotin compounds, compoundsthat will have uses as biotin agonists, antagonists, and biotincarboxylase inhibitors. The acyl halide “handle” on Compound 3 is aversatile tool allowing for ready reaction with a wide variety ofnucleophiles. Currently we are preparing and testing a variety ofcongeners of 1 as inhibitors of biotin carboxylase, for example,Compound 1+AMP, Compound 1+ADP, and Compound 1+ATP. These compoundscould serve as leads for novel antihyperlipidemic drugs and highlyspecific biodegradable herbicides, based on biotin carboxylase's role infatty acid biosynthesis. A different congener may (or may not) be abetter analog of the transition state, and thus could show betterinhibition of the enzyme. For example, the compounds (includingCompounds 1 and 3) will be used to inhibit proteases such as HIVprotease.

For example, experiments are currently underway to confirm theeffectiveness of Compounds 1 and 3 as inhibitors of HIV protease.Varying concentrations of these Compounds will be tested (starting at 8mM, and increasing or decreasing by powers of 2) in a standard HIVprotease inhibition assay to characterize the Compounds' effects on theenzyme. In particular, we will use the HIV protease activity assay ofBACHEM Bioscience (King of Prussia, Pa.) based on recombinant HIVprotease 1, with acetyl pepstatin as an inhibitor for control studies,in accordance with the manufacturer's recommendations. See Product No.H-9040 Analytical Data Sheet, BACHEM Bioscience (1999?).

A rigorous characterization of the inhibition of biotin carboxylase by 1and its derivatives is also in progress in our laboratories. Preliminaryresults of these enzymology studies may be found in C. Blanchard et al.,“Inhibition of Biotin Carboxylase by a Reaction Intermediate Analog:Implications for the Kinetic Mechanism” (unpublished manuscript, 1999).The analog showed competitive inhibition versus ATP with a slopeinhibition constant of 8 mM. Noncompetitive inhibition was found for theanalog versus biotin. Phosphonoacetate exhibited competitve inhibitionwith respect to ATP and noncompetitve inhibition versus bicarbonate.Multiple inhibition studies indicated that the binding of the reactionintermediate analog and ADP or AMP to biotin carboxylase was mutuallyexclusive. Biotin was found to be a noncompetitive substrate inhibitorof biotin carboxylase. These data were interpreted as biotin carboxylasehaving an ordered addition of substrates, with ATP binding first,followed by bicarbonate, with biotin binding last.

When used as an HIV protease inhibitor in vivo, a compound in accordancewith the present invention may be administered to a patient by anysuitable means, including oral, intravenous, parenteral, subcutaneous,intrapulmonary, and intranasal administration. Parenteral infusionsinclude intramuscular, intravenous, intraarterial, or intraperitonealadministration. The compound may also be administered transdermally, forexample in the form of a slow-release subcutaneous implant, or orally inthe form of capsules, powders, or granules. It may also be administeredby inhalation.

Pharmaceutically acceptable carrier preparations for parenteraladministration include sterile, aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's, or fixed oils. The active therapeutic ingredient maybe mixed with excipients that are pharmaceutically acceptable and arecompatible with the active ingredient. Suitable excipients includewater, saline, dextrose, glycerol and ethanol, or combinations thereof.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers, such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, inertgases, and the like.

The form may vary depending upon the route of administration. Forexample, compositions for injection may be provided in the form of anampule, each containing a unit dose amount, or in the form of acontainer containing multiple doses.

The compound maybe formulated into therapeutic compositions aspharmaceutically acceptable salts. These salts include acid additionsalts formed with inorganic acids, for example hydrochloric orphosphoric acid, or organic acids such as acetic, oxalic, or tartaricacid, and the like. Salts also include those formed from inorganic basessuch as, for example, sodium, potassium, ammonium, calcium or ferrichydroxides, and organic bases such as isopropylamine, trimethylamine,histidine, procaine and the like.

Controlled delivery may be achieved by admixing the active ingredientwith appropriate macromolecules, for example, polyesters, polyaminoacids, polyvinyl pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, prolamine sulfate, or lactide/glycolidecopolymers. The rate of release of the active compound may be controlledby altering the concentration of the macromolecule.

Another method for controlling the duration of action comprisesincorporating the active compound into particles of a polymericsubstance such as a polyester, peptide, hydrogel, polylactide/glycolidecopolymer, or ethylenevinylacetate copolymers. Alternatively, an activecompound may be encapsulated in microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization, for example,by the use of hydroxymethylcellulose or gelatin-microcapsules orpoly(methylmethacrylate) microcapsules, respectively, or in a colloiddrug delivery system. Colloidal dispersion systems include macromoleculecomplexes, nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes.

A problem with existing uses of biotin in biotin/avidin orbiotin/streptavidin systems is that the strong affinity of these pairsmakes it difficult to isolate a protein or nucleic acid bound to onemember of the pair. Modification of the 1′-N of biotin may lessen thebinding avidity of biotin to avidin and streptavidin, thereby making iteasier to isolate the protein or nucleic acid.

The complete disclosures of all references cited in this specificationare hereby incorporated by reference. Also incorporated by reference arethe following publications of the inventors' own work, which are notprior art to this application: D. Amspacher et al., “Synthesis of aReaction Intermediate Analog of Biotin-Dependent Carboxylases via aSelective Derivatization of Biotin,” manuscript submitted to OrganicLetters (1999); D. Amspacher et al., “Studies on the Catalytic Mechanismof Biotin,” Abstract, Steenbock Symposium (University of Wisconsin,Madison, May 28-31, 1998); D. Amspacher et al., “Synthesis andCharacterization of a Slow-Binding Inhibitor of Biotin Carboxylase,” pp.131-138 in P. Frey et al. (Eds.), Enzymatic Mechanisms (1999); D.Amspacher et al., “The Synthesis of a Slow-Binding Inhibitor of BiotinCarboxylase via a Selective Derivatization of Biotin,” Poster presentedat 216th American Chemical Society National Meeting (Boston, Aug. 23-27,1998); D. Amspacher et al., “The Synthesis of a Slow-Binding Inhibitorof Biotin Carboxylase via a Selective Derivatization of biotin,”Newsletter and Abstracts, 216th American Chemical Society NationalMeeting (Boston, Aug. 23-27, 1998).

In the event of an otherwise irreconcilable conflict, however, thepresent specification shall control.

We claim:
 1. A method for synthesizing a 1′-N-substituted biotincompound, said method comprising reacting phenylmethyl5-(6,8-diaza-6-(2-X-acetyl)-7-oxo-3-thiabicyclo[3.3.0]oct-2-yl)pentanoatewith a nucleophile, under reaction conditions favorable to nucleophilicsubstitution, wherein X is a halogen atom.
 2. A method as recited inclaim 1, wherein X is fluorine.
 3. A method as recited in claim wherein1, wherein X is chlorine.
 4. A method as recited in claim 1, wherein Xis bromine.
 5. A method as recited in claim 1, wherein X is iodine.
 6. A1′-N substituted biotin compound produced by the method of claim
 1. 7. A1′-N substituted biotin compound produced by the method of claim
 2. 8. A1′-N substituted biotin compound produced by the method of claim
 3. 9. A1′-N substituted biotin compound produced by the method of claim
 4. 10.A 1′-N substituted biotin compound produced by the method of claim 5.11. A method for inhibiting the activity of HIV protease, comprisingreacting the protease with an amount of a compound as recited in claim 6sufficient to reduce the activity of the protease to a statisticallysignificant degree.
 12. A method for inhibiting the activity of HIVprotease, comprising reacting the protease with an amount of a compoundas recited in claim 7 sufficient to reduce the activity of the proteaseto a statistically significant degree.
 13. A method for inhibiting theactivity of HIV protease, comprising reacting the protease with anamount of a compound as recited in claim 8 sufficient to reduce theactivity of the protease to a statistically significant degree.
 14. Amethod for inhibiting the activity of HIV protease, comprising reactingthe protease with an amount of a compound as recited in claim 9sufficient to reduce the activity of the protease to a statisticallysignificant degree.
 15. A method for inhibiting the activity of HIVprotease, comprising reacting the protease with an amount of a compoundas recited in claim 10 sufficient to reduce the activity of the proteaseto a statistically significant degree.